This project will investigate interactive signaling between the enteric nervous system (ENS), spinal sensory afferent innervation and enteric mast cells with guinea pig small intestine as the experimental model. The project tests a hypothesis, supported by publications from earlier funding cycles and new preliminary data, that a positive feed-forward signaling loop between spinal afferents, ENS neurons and enteric mast cells amplifies nociceptive and other forms of sensory input from the gut to the central nervous system. Neurophysiology of ENS-spinal afferent-mast cell interactions is basic for understanding visceral hypersensitivity and emerging recognition that functional abdominal pain involves co-morbidity of intestinal hypersensitivity with other pain syndromes (e.g., interstitial cystitis, prostatitis, vulvodynia and fibromyalgia). Primary objectives are to learn: a) which of the multiple morphological, electrophysiological and immunohistochemical neuronal subtypes in the ENS receive synaptic input from spinal afferents; b) kinds of synaptic potentials evoked by afferent stimulation and identification of neurotransmitters and receptors involved; c) how afferents innervate enteric mast cells to evoke release of mast cell mediators; d) the identity of the neurotransmitters released at afferent-mast cell junctions and the postjunctional receptors; e) how afferent-evoked release of mast cell mediators influences the electrophysiological and synaptic behavior of identified functional subclasses of neurons in the ENS. The specific aims are: Aim 1. Identification of the neurotransmitters released by intramural spinal afferents at their synapses with individually identified ENS neurons and the transmitters that evoke release of histamine and mast cell proteases form intramural mast cells in response to electrical or chemical activation of the afferents in intestinal preparations in vitro. Aim 2. Identification of the post synaptic/postjunctional receptors and receptor subtypes that mediate the actions of neurotransmitters released by spinal afferents at synapses with enteric neurons and identification of the receptor subtypes responsible for the release of histamine and mast cell proteases that we find during electrical or chemical activation of the afferents in pilot/feasibility studies. Aim 3. Identification of neurotransmitters released by motor neurons of the ENS at their junctions with neighboring mast cells and to identify the receptor subtypes for the transmitters, which activate the mast cells to release preformed and lipid membrane-derived paracrine signals (e.g., histamine, mast cell proteases and prostaglandins). PUBLIC HEALTH RELEVANCE: This 5-year project is a study of interactive signaling between the enteric nervous system (ENS), spinal sensory afferent nerves and enteric mast cells, with the guinea pig small intestine as the experimental model. The project tests a hypothesis, supported by publications from earlier funding cycles and new preliminary data, that a positive feed-forward signaling loop between spinal afferents, ENS neurons and enteric mast cells amplifies nociceptive and other forms of sensory input from the gut to the central nervous system. The information to be gained from this neurophysiological investigation of interactions between spinal afferents, ENS and enteric mast cells is basic for translational understanding of visceral hypersensitivity and the emerging recognition that functional abdominal pain can involve comorbidity of gut hypersensitivity with other pain syndromes elsewhere in the body (e.g., interstitial cystitis, prostatitis, vulvodynia, vulvar vestibulitis and fibromyalgia).